The present invention relates to single chain multivalent antibodies.
Antibodies are proteins belonging to a group of immunoglobulins elicited by the immune system in response to a specific antigen or substance which the body deems foreign. There are five classes of human antibodies, each class having the same basic structure. The basic structure of an antibody is a tetramer, or a multiple thereof, composed of two identical heterodimers each consisting of a light and a heavy chain. The light chain is composed of one variable (V) and one constant (C) domain, while a heavy chain is composed of one variable and three or more constant domains. The variable domains from both the light and heavy chain, designated V.sub.L and V.sub.H respectively, determine the specificity of an immunoglobulin, while the constant (C) domains carry out various effector functions.
Amino acid sequence data indicate that each variable domain comprises three complementarity determining regions (CDR) flanked by four relatively conserved framework regions (FR). The FR are thought to maintain the structural integrity of the variable region domain. The CDR have been assumed to be responsible for the binding specificity of individual antibodies and to account for the diversity of binding of antibodies.
As the basic structure of an antibody contains two heterodimers, antibodies are multivalent molecules. For example, the IgG classes have two identical antigen binding sites, while the pentameric IgM class has 10 identical binding sites.
Monoclonal antibodies having identical genetic parentage and binding specificity have been useful both as diagnostic and therapeutic agents. Monoclonal antibodies are routinely produced by hybridomas generated by fusion of mouse lymphoid cells with an appropriate mouse myeloma cell line according to established procedures. The administration of murine antibodies for in vivo therapy and diagnostics in humans is limited however, due to the human anti-mouse antibody response illicited by the human immune system.
Chimeric antibodies, in which the binding or variable regions of antibodies derived from one species are combined with the constant regions of antibodies derived from a different species, have been produced by recombinant DNA methodology. See, for example, Sahagen et al., J. Immunol. 137:1066-1074 (1986); Sun et al., Proc. Natl. Acad. Sci. USA, 82:214-218 (1987); Nishimura et al., Cancer Res. 47:999-1005 (1987); and Lie et al. Proc Natl. Acad. Sci. USA, 84:3439-3443 (1987) which disclose chimeric antibodies to tumor-associated antigens. Typically, the variable region of a murine antibody is joined with the constant region of a human antibody. It is expected that as such chimeric antibodies are largely human in composition, they will be substantially less immunogenic than murine antibodies.
Chimeric antibodies still carry the Fc regions which are not necessary for antigen binding, but constitute a major portion of the overall antibody structure which affects its pharmacokinetics. For the use of antibodies in immunotherapy or immunodiagnostics, is it desirable to have antibody-like molecules which localize and bind to the target tissue rapidly and for the unbound material to quickly clear from the body. Generally, smaller antibody fragments have greater capillary permeability and are more rapidly cleared from the body than whole antibodies.
Since it is the variable regions of light and heavy chains that interact with an antigen, single chain antibody fragments (scFvs) have been created with one V.sub.L and one V.sub.H, containing all six CDR's, joined by a peptide linker (U.S. Pat. No. 4,946,778) to create a V.sub.L -L-V.sub.H polypeptide, wherein the L stands for the peptide linker. A scFv wherein the V.sub.L and V.sub.H domains are orientated V.sub.H -L-V.sub.L is disclosed in U.S. Pat. No. 5,132,405.
As the scFvs have one binding site as compared to the minimum of two for complete antibodies, the scFvs have reduced avidity as compared to the antibody containing two or more binding sites.
It would therefore be advantageous to obtain constructions of scFvs having more than one binding site to enhance the avidity of the polypeptide, and retain or increase their antigen recognition properties. In addition it would be beneficial to obtain multivalent scFvs which are bispecific to allow for recognition of different epitopes on the target tissue, to allow for antibody-based recruitment of other immune effector functions, or allow antibody capture of a therapeutic or diagnostic moiety.